Oxidative inactivation of Escherichia coli glutamine synthetase (GS) involves the specific modification of a single histidine residue per subunit. This modification, which can be catalyzed by either mixed-function oxidase systems or by nonenzymatic systems, results in increased susceptibility to proteolytic atack. Several other enzymes have been found to be similarly inactivated. The purpose of this study is to determine whether oxidative modification of proteins may be involved in protein turnover in mammalian cells. In order to examine whether intracellular mammalian proteases recognize the oxidative modification, proteases which degrade oxidatively inactivated GS have been purified from mouse liver. Proteolysis was measured by the release of trichloroacetic acid soluble products from [14-C]GS. Lysosomal activity was largely due to cathepsin D, whereas nonlysosomal proteolysis was due to a serine protease and also to calcium-dependent proteolytic activity. These intracellular proteases were found to almost selectively degrade the oxidatively inactivated form of GS at physiological pH values, whereas trypsin showed only a 2 to 3-fold increase in proteolysis of the inactive GS. These observations suggest a possible role of oxidative inactivation in marking enzymes for intracellular degradation. Work will continue to examine the possible relationship between oxidative modification of proteins and the mechanism of protein degradation in mammalian cells.